EP0134307B1 - Preactivated surfaces of synthetic materials for immobilizing organochemical and biological substances, process for preparing them and their use - Google Patents

Preactivated surfaces of synthetic materials for immobilizing organochemical and biological substances, process for preparing them and their use Download PDF

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Publication number
EP0134307B1
EP0134307B1 EP83111144A EP83111144A EP0134307B1 EP 0134307 B1 EP0134307 B1 EP 0134307B1 EP 83111144 A EP83111144 A EP 83111144A EP 83111144 A EP83111144 A EP 83111144A EP 0134307 B1 EP0134307 B1 EP 0134307B1
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EP
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Prior art keywords
activation
immobilizing
coupling
synthetic materials
organochemical
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EP83111144A
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German (de)
French (fr)
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EP0134307A2 (en
EP0134307A3 (en
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André Dipl.-Chem. Gadow
W. Graham Wood
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HENNING BERLIN ANLAGEN GmbH
BRAHMS Diagnostica GmbH
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Henning Berlin GmbH Chemie- und Pharmawerk
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Priority to AT83111144T priority Critical patent/ATE42317T1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/808—Automated or kit

Definitions

  • the present invention relates to preactivated plastic surfaces for the immobilization of organic chemical and biological materials such as antigens, antibodies, allergens, tissues, enzymes, cofactors etc. Furthermore, the invention relates to a method for producing such preactivated plastic surfaces and their use.
  • Immobilized antibodies and antigens are often used in radio, enzyme, fluorescence and luminescence immunoassays. Histological tests use immobilized tissue.
  • the so-called RAST test radio allergy sorbent test
  • allergen diagnostics uses allergens immobilized on paper discs (e.g. birch pollen, etc.).
  • Immobilized enzymes are e.g. B. used in so-called enzyme reactors, but also immobilized enzyme substrates and cofactors are used in enzymatic tests, bound to solid supports.
  • the immobilization can take place on various solid phases such as glass, plastics or other homogeneous solids, using either the vessel wall (tubes or microtiter plates) or the surface of micro and macro particles (grains, spheres, disks etc.).
  • a popular solid for the immobilization of various organic chemical and biological materials is polystyrene, which can be coupled to micro and macro particles.
  • polylysine In addition to polylysine, other polyamines such as poly-L-arginine, poly-L-histidine or poly-L-ornithine have also been investigated, polylysine giving the best values; see. Maija Leinonen and Carl E. Frasch, Infection and Immunity, 1982, pp. 1203-1207 and Barry M. Gray, Journal of Immunological Methods, 28 (1979) pp. 187-192.
  • the object of the invention is to develop a method for preactivating plastic surfaces which is suitable for immobilizing organic-chemical and biological materials firmly and reproducibly on plastics without the known disadvantages of previously existing methods.
  • Extensive investigations regarding the optimization of the properties taking into account the various parameters such as temperature, reaction time, pH value and buffer system, have led to the surprising result that this object can be achieved by plastic surfaces that are coated with a polypeptide that consists of a defined copolymer consists of hydrophobic amino acids such as phenylalanine, valine, leucine etc. as well as amino acids with side chains which carry groups capable of activation and coupling (lysine, arginine, ornithine, cysteine, glutamic acid, aspartic acid etc.).
  • polypeptides such as poly-phenylalanine-lysine, poly-phenylalanine-glutamic acid, poly-leucine-lysine, poly-valine-cysteine, etc. Phenylalanine-lysine copolymers are particularly preferred.
  • the present invention accordingly relates to preactivated plastic surfaces which are coated with a polypeptide for immobilizing organic-chemical and biological materials, characterized in that the polypeptide is a defined copolymer of hydrophobic amino acids and amino acids with side chains capable of activation and coupling.
  • the coating can be carried out by treating the plastic surfaces with a solution or suspension of the polypeptide and then washing them. An activation step and the coupling can then follow.
  • the phenyl-lysine copolymer used preferably has a molar ratio of phenylalanine to lysine of 1: 1. In principle, however, polymers in other ratios (2: 1, 1: 2, etc.) can also be used successfully.
  • the molecular weight of the polypeptides should preferably be greater than 10,000. Polypeptides with a molecular weight of 30,000 to over 200,000 are particularly suitable.
  • polypeptides are already on the market and are, for example, available from Miles-Yeda Ltd. (Kiryat Weizmann Rehovot, Israel) or SIGMA Chemie GmbH, Kunststoff. Low molecular weight polypeptides are generally readily water-soluble; higher molecular weight ones are generally water-soluble in heat or can still be easily suspended.
  • the preactivatable plastic surfaces are brought into contact with such solutions or suspensions, preferably at room temperature or refrigerator temperature, for a few hours and then washed.
  • the plastic surfaces cover themselves with an almost constant thin layer of the polypeptide.
  • the hydrophobic portions of the polypeptide in the case of polyphenylalanine lysine, this is the phenylalanine portion
  • the portions bearing the activatable groups for example the lysine side chain with the terminal amino group, which are mostly hydrophilic, point outwards.
  • These groups can be activated via reactions described in the literature and following this activation, the coupling of the organic chemical and biological materials take place directly. The activation necessary before the coupling can take place, for example, with the aid of coupling reagents.
  • Coupling reagents suitable for activation are, for example, glutardialdehyde, cyanogen bromide, hydrazines, bisepoxiranes, divinylsulfones, epichlorohydrin, benzoquinones, trichloros-triazines, isothiocyanates, arylamines and phenylhydrazines for direct coupling.
  • the resulting bond types are, for example, Michael adducts and Schiff bases, cyanate esters, triazinyls, ethers, imidocarbonates, amides, mixed anhydrides, alkylamines, esters.
  • Reagents such as EEDQ (N - ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) carbodiimides such as dicyclohexylcarbodiimide, 1-cyclohexyl-3- (2-morpholinyl- (4) -ethyl) carbodiimide-methyl- are suitable for indirect coupling p-toluenesulfonate, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimides.
  • Heterobifunctional reagents such as MBS (m - maleimidobenzoyl-N-hydroxysuccinimide ester) and others are also suitable.
  • Phenylalanine-lysine copolymers thus belong to a class of substances which, due to their properties, can be used in general to preactivate plastic surfaces to which components capable of coupling such as antigens, antibodies, allergens, tissues, enzymes, substrates, cofactors etc. are to be bound.
  • the pre-activation works reliably and consistently regardless of which reagents are used for the final coupling of the material to be immobilized.
  • plastic surfaces preactivated according to the invention and processes for their production and the coupling of different materials are explained in more detail:
  • Coupling time 38 h at 4 ° C, 125 1-T 4 was offered in excess, dissolved in 0.01 mol / l phosphate buffer pH 8.5.
  • the ratio of the specific and non-specific binding to the total activity offered is given after a reaction time of 6 h at room temperature. Radioactiv-labeled 125 1-TBG (thyroxine-binding globulin) was used to measure the non-specific binding.
  • Polystyrene tubes are filled with the same solution as in Example 1, left to stand overnight and rinsed 1x with 0.15 mol (I NaCl in demin. H 2 0 and 2x with demin. H 2 0.
  • the tubes treated in this way are washed with the one in Example 1 described pentane-1,5-dial solution, reacted for 30 minutes at room temperature and washed accordingly
  • the tubes treated in this way are then immediately suitable for firmly binding organic-chemical and biological materials with amino groups capable of coupling.
  • Example 2 The individual wells of a microtiter plate are filled with the solution described in Example 1 and further treated as in Example 2.
  • the plate binds biological material.
  • Tubes made of polyethylene and polypropylene are preactivated as described in Example 2.
  • the tubes treated in this way like polystyrene tubes, are suitable for firmly binding organic chemical and biological materials with amino groups capable of coupling.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Compounds Of Unknown Constitution (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

Plastics surfaces are preactivated for immobilizing organo-chemical and biologic materials by coating the surfaces with a polypeptide which consists of a defined composition of hydrophobic amino acids and amino acids having side chains capable of activation and coupling. After activation of the plastics surfaces having been coated in this manner with various coupling reagents, it is possible to firmly fix organo-chemical and biologic materials. The materials having been immobilized in this manner are extremely stable to various reaction conditions.

Description

Gegenstand der vorliegenden Erfindung sind voraktivierte Kunststoffoberflächen zur Immobilisierung von organisch-chemischen und biologischen Materialien wie Antigenen, Antikörpern, Allergenen, Geweben, Enzymen, Cofaktoren etc. Weiterhin betrifft die Erfindung ein Verfahren zur Herstellung solcher voraktivierter Kunststoffoberflächen sowie ihre Verwendung.The present invention relates to preactivated plastic surfaces for the immobilization of organic chemical and biological materials such as antigens, antibodies, allergens, tissues, enzymes, cofactors etc. Furthermore, the invention relates to a method for producing such preactivated plastic surfaces and their use.

Bei vielen immunologischen, histologischen und enzymatischen Methoden ist es notwendig und zur Vereinfachung der Verfahren zweckmäßig, entsprechende Komponenten des Tests an festen Trägern zu immobilisieren. Bei Radio-, Enzym-, Fluoreszenz- und Lumineszenz-Immunoassays werden häufig immobilisierte Antikörper und Antigene verwendet. Bei histologischen Tests verwendet man immobilisiertes Gewebe. Der sog. RAST-Test (Radio-Allergo-Sorbens-Test) für die Allergendiagnostik verwendet an Papierscheibchen immobilisierte Allergene (z. B. Birkenpollen etc.). Immobilisierte Enzyme werden z. B. in sog. Enzymreaktoren verwendet, aber auch immobilisierte Enzym-Substrate und Cofaktoren werden in enzymatischen Tests, gebunden an feste Träger verwendet. Die Immobilisierung kann an verschiedenen festen Phasen wie Glas, Kunststoffen oder sonstigen homogenen Festkörpern erfolgen, wobei entweder die Gefäßwand (Röhrchen bzw. Mikrotiterplatten) oder die Oberfläche von Mikro- und Makropartikeln (Körner, Kugeln, Scheibchen etc.) eingesetzt werden. Ein beliebter Festkörper für die Immobilisierung von verschiedenen organisch-chemischen und biologischen Materialien ist Polystyrol, wobei die Kopplung an Mikro und Makropartikel erfolgen kann.With many immunological, histological and enzymatic methods, it is necessary and appropriate to simplify the methods to immobilize corresponding components of the test on solid supports. Immobilized antibodies and antigens are often used in radio, enzyme, fluorescence and luminescence immunoassays. Histological tests use immobilized tissue. The so-called RAST test (radio allergy sorbent test) for allergen diagnostics uses allergens immobilized on paper discs (e.g. birch pollen, etc.). Immobilized enzymes are e.g. B. used in so-called enzyme reactors, but also immobilized enzyme substrates and cofactors are used in enzymatic tests, bound to solid supports. The immobilization can take place on various solid phases such as glass, plastics or other homogeneous solids, using either the vessel wall (tubes or microtiter plates) or the surface of micro and macro particles (grains, spheres, disks etc.). A popular solid for the immobilization of various organic chemical and biological materials is polystyrene, which can be coupled to micro and macro particles.

Zur besser reproduzierbaren Immobilisierung spezieller Antikörper wurden diese kovalent an Poly-Lysin gebunden, welches seinerseits an Polystyrolkugeln adsorbiert war.For more reproducible immobilization of special antibodies, these were covalently bound to poly-lysine, which in turn was adsorbed on polystyrene balls.

Außer Polylysin sind auch andere Polyamine wie Poly-L-arginine, Poly-L-histidine oder Poly-L-ornithine untersucht worden, wobei Polylysin die besten Werte ergab; vgl. Maija Leinonen und Carl E. Frasch, Infection and Immunity, 1982, S. 1203-1207 sowie Barry M. Gray, Journal of Immunological Methods, 28 (1979) S. 187-192.In addition to polylysine, other polyamines such as poly-L-arginine, poly-L-histidine or poly-L-ornithine have also been investigated, polylysine giving the best values; see. Maija Leinonen and Carl E. Frasch, Infection and Immunity, 1982, pp. 1203-1207 and Barry M. Gray, Journal of Immunological Methods, 28 (1979) pp. 187-192.

Alle bisher untersuchten Kunststoffoberflächen zur Immobilisierung von Antikörpern oder Antigenen weisen noch erhebliche Nachteile auf, da sie entweder zu nicht reproduzierbaren Werten führen oder aber zu unerwünschten schlecht kontrollierbaren Auswaschreaktionen oder unspezifischen Veränderungen der Antikörper oder Antigene.All previously investigated plastic surfaces for the immobilization of antibodies or antigens still have considerable disadvantages, since they either lead to non-reproducible values or to undesirable wash-out reactions which are difficult to control or unspecific changes in the antibodies or antigens.

Die Erfindung hat sich die Aufgabe gestellt, ein Verfahren zur Voraktivierung von Kunststoffoberflächen zu entwickeln, das geeignet ist, organisch-chemische und biologische Materialien fest und reproduzierbar ohne die bekannten Nachteile bisher bestehender Verfahren an Kunststoffen zu immobilisieren. Umfangreiche und die verschiedensten Parameter wie Temperatur, Reaktionsdauer, pH-Wert und Puffersystem berücksichtigende Untersuchungen bezüglich der Optimierung der Eigenschaften haben zu dem überraschenden Ergebnis geführt, daß diese Aufgabe gelöst werden kann durch Kunststoffoberflächen, die mit einem Polypeptid beschichtet werden, das aus einem definierten Copolymerisat von hydrophoben Aminosäuren wie Phenylalanin, Valin, Leucin usw. besteht sowie aus Aminosäuren mit Seitenketten die zur Aktivierung und Kopplung befähigte Gruppen tragen (Lysin, Arginin, Ornithin, Cystein, Glutaminsäure, Asparaginsäure usw.).The object of the invention is to develop a method for preactivating plastic surfaces which is suitable for immobilizing organic-chemical and biological materials firmly and reproducibly on plastics without the known disadvantages of previously existing methods. Extensive investigations regarding the optimization of the properties, taking into account the various parameters such as temperature, reaction time, pH value and buffer system, have led to the surprising result that this object can be achieved by plastic surfaces that are coated with a polypeptide that consists of a defined copolymer consists of hydrophobic amino acids such as phenylalanine, valine, leucine etc. as well as amino acids with side chains which carry groups capable of activation and coupling (lysine, arginine, ornithine, cysteine, glutamic acid, aspartic acid etc.).

Typische Vertreter dieser Substanzen sind somit Polypeptide wie Poly-Phenylalanin-Lysin, Poly- Phenylalanin-Glutaminsäure, Poly-Leucin-Lysin, Poly-Valin-Cystein usw. Besonders bevorzugt sind Phenylalanin-Lysin-Copolymere.Typical representatives of these substances are thus polypeptides such as poly-phenylalanine-lysine, poly-phenylalanine-glutamic acid, poly-leucine-lysine, poly-valine-cysteine, etc. Phenylalanine-lysine copolymers are particularly preferred.

Gegenstand der vorliegenden Erfindung sind demnach voraktivierte Kunststoffoberflächen, die mit einem Polypeptid beschichtet sind zur Immobilisierung von organisch-chemischen und biologischen Materialien, dadurch gekennzeichnet, daß das Polypeptid ein definiertes Copolymerisat von hydrophoben Aminosäuren und Aminosäuren mit zur Aktivierung und Kopplung befähigten Seitenketten ist.The present invention accordingly relates to preactivated plastic surfaces which are coated with a polypeptide for immobilizing organic-chemical and biological materials, characterized in that the polypeptide is a defined copolymer of hydrophobic amino acids and amino acids with side chains capable of activation and coupling.

Die Beschichtung kann erfindungsgemäß dadurch erfolgen, daß man die Kunststoffoberflächen mit einer Lösung oder Suspension des Polypeptids behandelt und anschließend wäscht. Im Anschluß kann dann ein Aktivierungsschritt und die Kopplung folgen.According to the invention, the coating can be carried out by treating the plastic surfaces with a solution or suspension of the polypeptide and then washing them. An activation step and the coupling can then follow.

Das verwendete Phenyl-Lysin-Copolymer weist vorzugsweise ein Molverhältnis von Phenylalanin zu Lysin von 1 : 1 auf. Prinzipiell sind jedoch auch Polymere in anderen Verhältnissen (2 : 1, 1 : 2, usw.) mit Erfolg einsetzbar.The phenyl-lysine copolymer used preferably has a molar ratio of phenylalanine to lysine of 1: 1. In principle, however, polymers in other ratios (2: 1, 1: 2, etc.) can also be used successfully.

Das Molekulargewicht der Polypeptide sollte vorzugsweise größer als 10.000 sein. Hervorragend geeignet sind Polypeptide mit einem Molekulargewicht von 30.000 bis über 200.000.The molecular weight of the polypeptides should preferably be greater than 10,000. Polypeptides with a molecular weight of 30,000 to over 200,000 are particularly suitable.

Derartige Polypeptide sind bereits im Handel und werden beispielsweise von den Firmen Miles-Yeda Ltd. (Kiryat Weizmann Rehovot, Israel) oder SIGMA Chemie GmbH, München angeboten. Niedermolekulare Polypeptide sind im allgemeinen leicht wasserlöslich, höhermolekulare sind im allgemeinen in der Wärme wasserlöslich oder noch gut suspendierbar.Such polypeptides are already on the market and are, for example, available from Miles-Yeda Ltd. (Kiryat Weizmann Rehovot, Israel) or SIGMA Chemie GmbH, Munich. Low molecular weight polypeptides are generally readily water-soluble; higher molecular weight ones are generally water-soluble in heat or can still be easily suspended.

Die voraktivierbaren Kunststoffoberflächen werden mit derartigen Lösungen oder Suspensionen vorzugsweise bei Raumtemperatur oder Kühlschranktemperatur einige Stunden in Berührung gebracht und danach gewaschen. Die Kunststoffoberflächen überziehen sich dabei mit einer nahezu konstanten dünnen Schicht des Polypeptids. Die hydrophoben Anteile des Polypeptides (im Falle von Poly-PhenylalaninLysin ist dies der Phenylalaninanteil) lagern sich dabei direkt auf der Kunststoffoberfläche an und die die aktivierbaren Gruppen tragenden Anteile (beispielsweise also die Lysin-Seitenkette mit der endständigen Aminogruppe), die meistens hydrophil sind, zeigen nach außen. Diese Gruppen können über in der Literatur zahlreich beschriebene Reaktionen aktiviert werden und im Anschluß an diese Aktivierung kann die Kopplung der organisch-chemischen und biologischen Materialien direkt erfolgen. Die vor der Kopplung notwendige Aktivierung kann beispielsweise mit Hilfe von Kopplungsreagenzien erfolgen.The preactivatable plastic surfaces are brought into contact with such solutions or suspensions, preferably at room temperature or refrigerator temperature, for a few hours and then washed. The plastic surfaces cover themselves with an almost constant thin layer of the polypeptide. The hydrophobic portions of the polypeptide (in the case of polyphenylalanine lysine, this is the phenylalanine portion) are deposited directly on the plastic surface and the portions bearing the activatable groups (for example the lysine side chain with the terminal amino group), which are mostly hydrophilic, point outwards. These groups can be activated via reactions described in the literature and following this activation, the coupling of the organic chemical and biological materials take place directly. The activation necessary before the coupling can take place, for example, with the aid of coupling reagents.

Zur Aktivierung geeignete Kopplungsreagenzien sind beispielsweise für die direkte Kopplung Glutardialdehyd, Bromcyan, Hydrazine, Bisepoxirane, Divinylsulfone, Epichlorhydrin, Benzochinone, Trichloros-Triazine, Isothiocyanate, Arylamine und Phenylhydrazine. Die dabei entstehenden Bindungstypen sind beispielsweise Michael Addukte und Schiff'sche Basen, Cyanat-Ester, Triazinyle, Ether, Imidocarbonate, Amide, gemischte Anhydride, Alkylamine, Ester. Für die indirekte Kopplung kommen Reagenzien in Frage wie EEDQ (N - Ethoxycarbonyl-2-ethoxy-1,2-dihydrochinolin) Carbodiimide wie Dicyclohexylcarbodiimid, 1-Cyclohexyl-3-(2-morpholinyl-(4)-ethyl) carbodiimid-methyl-p-toluol-sulfonat, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimid hydrochlorid und N - Hydroxysuccinimide. Weiterhin kommen heterobifunktionelle Reagenzien in Frage wie MBS (m - Maleimidobenzoyl-N-hydroxysuccinimidester) und andere.Coupling reagents suitable for activation are, for example, glutardialdehyde, cyanogen bromide, hydrazines, bisepoxiranes, divinylsulfones, epichlorohydrin, benzoquinones, trichloros-triazines, isothiocyanates, arylamines and phenylhydrazines for direct coupling. The resulting bond types are, for example, Michael adducts and Schiff bases, cyanate esters, triazinyls, ethers, imidocarbonates, amides, mixed anhydrides, alkylamines, esters. Reagents such as EEDQ (N - ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) carbodiimides such as dicyclohexylcarbodiimide, 1-cyclohexyl-3- (2-morpholinyl- (4) -ethyl) carbodiimide-methyl- are suitable for indirect coupling p-toluenesulfonate, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimides. Heterobifunctional reagents such as MBS (m - maleimidobenzoyl-N-hydroxysuccinimide ester) and others are also suitable.

Vergleichende Untersuchungen mit anderen voraktivierten Oberflächen haben ergeben, daß erfindungsgemäß immobilisierte organisch-chemische und biologische Materialien besonders fest und vor allem mit ausgezeichneter Präzision und Reproduzierbarkeit an die Oberfläche gebunden werden, ohne daß es zu den bei den bestehenden Verfahren bekannten unspezifischen Reaktionen und Auswaschungsphänomen während späterer Verfahrensschritte kommt. Die Anwendung von sog. Detergenzien wie Tween 20 (Polyoxyethylensorbitanmonolaurat) und Triton X 100 (Polyethylenether) zur Wegwaschung von in späteren Verfahrensschritten unspezifisch gebundenen Komponenten ist problemlos möglich, ohne daß die spezifisch gekoppelten Anteile entfernt werden. Dies war bei den bestehenden rein adsorbtiven Immobilisierungsverfahren unter Verwendung von beispielsweise Polystyrol nicht möglich. Diese Vorteile wurden bei einer Vielzahl erfindungsgemäß immobilisierter organisch-chemischer und biologischer Materialien beobachtet, so daß erfindungsgemäß voraktivierte Kunststoffoberflächen bei den verschiedensten Anwendungsgebieten eingesetzt werden können.Comparative studies with other preactivated surfaces have shown that organochemical and biological materials immobilized according to the invention are bound to the surface particularly firmly and, above all, with excellent precision and reproducibility, without causing the unspecific reactions and leaching phenomenon known from the existing processes during later Procedural steps coming. The use of so-called detergents such as Tween 20 (polyoxyethylene sorbitan monolaurate) and Triton X 100 (polyethylene ether) for washing away components which were unspecifically bound in later process steps is possible without removing the specifically coupled portions. This was not possible with the existing purely adsorbent immobilization processes using, for example, polystyrene. These advantages have been observed in a large number of organic-chemical and biological materials immobilized according to the invention, so that plastic surfaces preactivated according to the invention can be used in a wide variety of fields of application.

Phenylalanin-Lysin-Copolymere gehören somit zu einer Substanzklasse die aufgrund ihrer Eigenschaften ganz allgemein zur Voraktivierung von Kunststoffoberflächen verwendet werden können, an die zur Kopplung befähigte Komponenten wie Antigene, Antikörper, Allergene, Gewebe, Enzyme, Substrate, Cofaktoren usw. gebunden werden sollen. Die Voraktivierung wirkt zuverlässig und gleichbleibend unabhängig davon mit welchen Reagenzien die endgültige Kopplung des zu immobilisierenden Materials erfolgt.Phenylalanine-lysine copolymers thus belong to a class of substances which, due to their properties, can be used in general to preactivate plastic surfaces to which components capable of coupling such as antigens, antibodies, allergens, tissues, enzymes, substrates, cofactors etc. are to be bound. The pre-activation works reliably and consistently regardless of which reagents are used for the final coupling of the material to be immobilized.

Erstmals beobachtet und beschrieben wurden diese überraschend guten Eigenschaften bei der Voraktivierung von Polystyrolkugeln mit Phenylalanin-Lysin-Copolymeren, Aktivierung mittels Glutardialdehyd und Immobilisierung von Antikörpern in der deutschen Patentanmeldung P-3 327 327.8. Es hat sich gezeigt, daß es sich hierbei um ein allgemeines und breit anwendbares Prinzip handelt. Als Kunststoffe kommen praktisch alle hydrophoben Kunststoffe in Frage wie Polystyrol, Polyethylen, Polypropylen etc.These surprisingly good properties were observed and described for the first time in the preactivation of polystyrene spheres with phenylalanine-lysine copolymers, activation by means of glutardialdehyde and immobilization of antibodies in the German patent application P-3 327 327.8. It has been shown that this is a general and widely applicable principle. Virtually all hydrophobic plastics, such as polystyrene, polyethylene, polypropylene, etc., are suitable as plastics.

In den nachfolgenden Beispielen sind erfindungsgemäß voraktivierte Kunststoffoberflächen sowie Verfahren zu ihrer Herstellung und der Kopplung verschiedener Materialien näher erläutert:In the following examples, plastic surfaces preactivated according to the invention and processes for their production and the coupling of different materials are explained in more detail:

Beispiel 1example 1

  • a) Immobilisierung nach Voraktivierunga) Immobilization after pre-activation
  • 5 mg Polyphenylalanin-Lysin der Firma Sigma (MG 40 KD) oder Miles-Yeda (MG 30KD) werden unter leichtem Erwärmen in insgesamt 200 ml entmineralisiertem Wasser gelöst und auf 1000 Polystyrolkugeln (Precision plastic balls, Chicago) vom Durchmesser 6,4 mm gegossen. Es wird ca. 30 min. bei Raumtemperatur unter gelegentlichem sanften Schütteln stehengelassen und dann 24 h bei Kühlschranktemperatur (4°) stehen gelassen. Die Kugeln werden dann einmal durch Überschichten mit einer 0,15 mol/1 Kochsalzlösung und zweimal mit demineralisiertem Wasser gewaschen. Die so beschichteten Polystyrolkugeln können nun direkt mit einem Kopplungsreagenz umgesetzt werden, beispielsweise mit 0,5 Volumen-o/o Pentan-1,5-dial (Glutardialdehyd) in demineralisiertem Wasser für 30 min. bei Raumtemperatur. Nach dem letztgenannten Aktivierungsschritt können aminogruppentragende organisch-chemische und biologische Materialien direkt gekoppelt werden. Es werden im allgemeinen Mengen von 1 - 5 pg pro Polystyrolkugel angeboten. Die Immobilisierung von Antikörpern ist in den Beispielen der deutschen Patentanmeldung P 33 27 327.8 beschrieben.5 mg of polyphenylalanine-lysine from Sigma (MG 40 KD) or Miles-Yeda (MG 30KD) are dissolved in a total of 200 ml of demineralized water with gentle heating and poured onto 1000 polystyrene balls (Precision plastic balls, Chicago) with a diameter of 6.4 mm . It will take approx. 30 min. Allow to stand at room temperature with occasional gentle shaking and then leave to stand at refrigerator temperature (4 °) for 24 hours. The balls are then washed once by overlaying with 0.15 mol / 1 saline solution and twice with demineralized water. The polystyrene spheres coated in this way can now be reacted directly with a coupling reagent, for example with 0.5% by volume pentane-1,5-dial (glutardialdehyde) in demineralized water for 30 min. at room temperature. After the last-mentioned activation step, organic-chemical and biological materials carrying amino groups can be coupled directly. Quantities of 1-5 pg per polystyrene ball are generally available. The immobilization of antibodies is described in the examples of German patent application P 33 27 327.8.
b) Vergleichende Untersuchungenb) Comparative studies

Tabelle 1 und 2 zeigen die Stabilität und Bindungseigenschaften von nach Beispiel 1a immobilisierten T4 (Thyroxin) sowie Antikörpern gegen h-TSH (Thyrotropin), und vergleichen sie mit den Eigenschaften einer herkömmlichen Immobilisierung. Vergleichende Übersicht der Immobilisierung von radioaktiv-markiertem 1251-T4 (Thyroxin) an Polystyrolkugeln

  • a) rein adsorbtiv,
  • b) nach Behandlung der Polystyrolkugeln mit Glutardialdehyd sowie
  • c) nach erfindungsgemäßer Beschichtung mit Phenylalanin-Lysin-Copolymer und anschließender Aktivierung mit Glutardialdehyd.
Tables 1 and 2 show the stability and binding properties of T 4 (thyroxine) immobilized according to Example 1a and antibodies against h-TSH (thyrotropin), and compare them with the properties of a conventional immobilization. Comparative overview of the immobilization of radioactively labeled 125 1-T 4 (thyroxine) on polystyrene balls
  • a) purely adsorbent,
  • b) after treatment of the polystyrene balls with glutardialdehyde and
  • c) after coating according to the invention with phenylalanine-lysine copolymer and subsequent activation with glutardialdehyde.

Kopplungsdauer 38 h bei 4°C, 1251-T4 wurde im Überschuß angeboten, gelöst in 0,01 mol/I Phosphatpuffer pH 8,5.

Figure imgb0001
Coupling time 38 h at 4 ° C, 125 1-T 4 was offered in excess, dissolved in 0.01 mol / l phosphate buffer pH 8.5.
Figure imgb0001

Vergleichende Übersicht der Bindung von radioaktiv-markiertem, Antikörper gegen humanes Thyrotropin, die unter optimierten Bedingungen

  • a) rein adsorptiv
  • b) nach Behandlung der Polystyrolkugeln mit Glutardialdehyd sowie
  • c) nach erfindungsgemäßer Beschichtung mit Phenylalanin-Lysin-Copolymer und anschließender Aktivierung mit Glutardialdehyd an Polystyrolkugeln gebunden wurden.
Comparative overview of binding of radio-labeled, antibodies against human thyrotropin, under optimized conditions
  • a) purely adsorptive
  • b) after treatment of the polystyrene balls with glutardialdehyde and
  • c) after coating according to the invention with phenylalanine-lysine copolymer and subsequent activation with glutardialdehyde were bound to polystyrene balls.

Angegeben ist das Verhältnis der spezifischen und unspezifischen Bindung zur angebotenen Totalaktivität nach einer Reaktionszeit von 6 h bei Raumtemperatur. Zur Messung der unspezifischen Bindung wurde radioaktlv-markiertes 1251-TBG (Thyroxinbindendes Globulin) verwendet.

Figure imgb0002
The ratio of the specific and non-specific binding to the total activity offered is given after a reaction time of 6 h at room temperature. Radioactiv-labeled 125 1-TBG (thyroxine-binding globulin) was used to measure the non-specific binding.
Figure imgb0002

Beispiel 2Example 2

Polystyrolröhrchen werden mit der gleichen Lösung wie in Beispiel 1 gefüllt, über Nacht stehengelassen und 1x mit 0,15 mol(I NaCI in demin. H20 sowie 2x mit demin. H20 ausgespült. Die so behandelten Röhrchen werden mit der im Beispiel 1 beschriebenen Pentan-1,5-dial-Lösung gefüllt, 30 min. bei Raumtemperatur zur Reaktion gebracht und entsprechend gewaschen. Die so behandelten Röhrchen sind danach unmittelbar geeignet, organisch-chemische und biologische Materialien mit zur Kopplung befähigten Aminogruppen fest zu binden.Polystyrene tubes are filled with the same solution as in Example 1, left to stand overnight and rinsed 1x with 0.15 mol (I NaCl in demin. H 2 0 and 2x with demin. H 2 0. The tubes treated in this way are washed with the one in Example 1 described pentane-1,5-dial solution, reacted for 30 minutes at room temperature and washed accordingly The tubes treated in this way are then immediately suitable for firmly binding organic-chemical and biological materials with amino groups capable of coupling.

Beispiel 3Example 3

Die einzelnen Vertiefungen einer Mikrotiterplatte werden mit der in Beispiel 1 beschriebenen Lösung gefüllt und wie in Beispiel 2 weiterbehandelt. Die Platte bindet biologisches Material.The individual wells of a microtiter plate are filled with the solution described in Example 1 and further treated as in Example 2. The plate binds biological material.

Beispiel 4Example 4

100 g Polystyrol in Mikropartikelform (Dow-Latex, Serva) werden mit der Lösung aus Beispiel 1 überschichtet. Die Weiterreaktion erfolgt in analoger Weise. Zur Waschung wird filtriert.100 g of polystyrene in the form of microparticles (Dow latex, Serva) are covered with the solution from Example 1. The further reaction takes place in an analogous manner. Filter for washing.

Bei der Aktivierung mit Glutardialdehyd und der anschließenden Kopplung von aminogruppentragenden Substanzen bilden sich Schiff'sche Basen, die unter Verwendung von Natriumborhydrid oder Natriumcyanoborhydrid reduziert werden können. Die erfindungsgemäße Beschichtung übersteht solche Behandlungen ohne Beeinträchtigungen.On activation with glutardialdehyde and the subsequent coupling of amino-bearing substances, Schiff bases are formed, which can be reduced using sodium borohydride or sodium cyanoborohydride. The coating according to the invention survives such treatments without impairments.

Beispiel 5Example 5

Röhrchen aus Polyethylen und Polypropylen werden wie im Beispiel 2 beschrieben voraktiviert. Die so behandelten Röhrchen sind in gleicher Weise wie Polystyrolröhrchen geeignet, organisch-chemische und biologische Materialien mit zur Kopplung befähigten Aminogruppen fest zu binden.Tubes made of polyethylene and polypropylene are preactivated as described in Example 2. The tubes treated in this way, like polystyrene tubes, are suitable for firmly binding organic chemical and biological materials with amino groups capable of coupling.

Claims (6)

1. Pre-activated surfaces of synthetic materials coated with a polypeptide for immobilizing organochemical and biological materials, characterized in that the polypeptide is a defined copolymer of hydrophobic amino acids and amino acids having side chains capable of activation and coupling.
2. Pre-activated surfaces of synthetic materials according to claim 1, characterized in that the surfaces have been coated with phenylalanine-lysine copolymer.
3. A process for the pre-activation surfaces of synthetic materials for immobilizing organochemical and biological materials, characterized in that the surface of the synthetic material is treated with a solution or suspension of polypeptide which is a defined copolymer of hydrophobic amino acids and amino acids having side chains capable of activation and coupling, and is subsequently washed.
4. The process according to claim 3, characterized in that a phenylalanine-lysine copolymer is used as the polypeptide.
5. Use of pre-activated surfaces of synthetic materials according to claims 1 and 2 for immobilizing organochemical and biological materials.
6. Use of pre-activated surfaces of synthetic materials according to claim 5, characterized in that antibodies or antigens are immobilized as biological material.
EP83111144A 1983-07-29 1983-11-08 Preactivated surfaces of synthetic materials for immobilizing organochemical and biological substances, process for preparing them and their use Expired EP0134307B1 (en)

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